Lyme disease is a common tickborne infection of the northern hemisphere's temperature latitudes. The clinical features and epidemiology of Lyme disease have been well-characterized, and the etiologic agent, the spirochete Borrelia burgdorferi, has been isolated (reviewed by Steere, 1989). Borrelia burgdorferi enters the host's vascular system from the tick bite site and then is distributed to different organs and tissues, including the brain and joint synovium. In these different tissues the microorganism can persist for months to years. The properties of Borrelia burgdorferi that confer invasiveness in the human and other mammalian hosts have yet to be completely identified, although the flagellum has been implicated in pathogenicity.
Diagnosis of Lyme disease is complicated by the fact that the disease may mimic several other disorders, many of which are not infectious and, therefore, not ameliorated by antibiotics. A challenge for physicians is to identify cases of pauciarticular arthritis, radiculopathy, or extreme chronic fatigue as Lyme disease. If the clinical impression is confirmed by specific diagnostic assays, appropriate antimicrobial therapy may reverse long-standing pathologic changes. Unfortunately, physicians are often frustrated in this process by the inadequacies of currently available diagnostic procedures.
Recovery of Borrelia burgdorferi from patients is possible and should be considered diagnostic. However, the medium is expensive to keep stocked, cultures require up to 4 weeks of incubation for routine detection of spirochetes, and the frequency of isolating bacteria from the blood of acutely ill patients is less than 30%. Consequently, cultivation for B. burgdorferi is only done in a few institutions.
Direct detection of the etiologic agent in tissue or body fluids has also been attempted for diagnosis. For early Lyme disease, when the dermatologic hallmark erythema chronicum migrans is present, a Warthin-Starry or modified Dieterle silver stain reveals spirochetes in one-half or more of skin biopsies obtained from the outer portion of lesions (Duray, 1987; Berger, et al., 1983). The microorganisms are comparatively sparse, however, and can be confused with normal skin structures by inexperienced laboratory personnel. Immunohistologic examination of tissue with monoclonal and polyclonal antibodies has also been used successfully to show the presence of borreliae, but there is less experience with this technique than with the silver stains. (Park, et al., 1986).
Cases not meeting the strict clinical and epidemiologic criteria for diagnosis have also been identified as Lyme disease by using a serologic test, usually an enzyme-linked immunosorbent assay (ELISA) or in-direct immunofluorescence assay (IFA). Although many public and private laboratories now offer either ELISA or IFA, the procedures for these assays have not yet been standardized. The antigen preparations and the "cut-off" values for a positive test vary among laboratories. Significant interlaboratory variations in test results and in interpretations of the same set of sera have been reported (Hedberg, et al., 1987).
Many present immunoassays use whole spirochetes or a crude sonicate of the cells. However, those assays suffer from complications resulting from cross-reactions with other spirochetes, especially Treponema palladium and the relapsing fever Borrelia species (Magnarelli and Anderson, 1988), and borderline or low-level positive titers in some patients with other rheumatologic or neurologic disorders. Because of increasing professional and lay awareness of Lyme disease, serologic testing is often requested to "rule out" the diagnosis. In this situation, the ratio of persons with false-positive reactions compared with those who have actual Borrelia burgdorferi infections will predictably rise. Thus, "seropositive" patients with disorders other than Lyme disease may be subjected to long and possibly hazardous courses of oral or parenteral antibiotics. A more specific diagnostic assay for B. burgdorferi is needed.
Although several investigators have suggested use of an immunoassay using a purified flagella protein antigen, there are problems with this approach. For one thing, it has recently been shown that a monoclonal antibody directed against the major flagella protein of Borrelia burgdorferi also recognizes human tissue, including myelin and Schwann cells from the peripheral nervous system (Sigal, et al., 1988; Aberer, et al., 1989). Autoantibodies against neural antigens have been observed in the serum of patients with Lyme disease. These findings suggest that autoreactive antibodies may complicate interpretation of immunoassays that use purified flagellar antigen or whole cell sonicates containing flagella antigen. Another problem with the use of preparations containing the flagellum by itself or in combination with other components is that there may be false positive reactions as a consequence of antigenic similarities between the flagella of borreliae and the flagella of other bacteria.
The discovery that flagellar antigens may induce formation of antibodies reactive with human neural tissue provides an additional problem with respect to development of a vaccine against Lyme Disease. International Patent Application No. WO 90/04411, published May 3, 1990 describes a method for preparing fractions of Borrelia burgdorferi in which the flagellar components are at least partially depleted. However, that method is somewhat time consuming and labor intensive. Inactivated whole cell Borrelia burgdorferi vaccines, such as those described in U.S. Pat. No. 4,721,617 and by Johnson, et al., 1986, comprise a relatively high proportion of the flagellar antigen and may thus induce an undesirable auto immune response. In addition, because flagellated Borrelia is virulent, the cells must be killed prior to administration, thus reducing immunogenicity. Because the flagellum is an important virulence factor of the organism, development of a flagellar-less strain could provide an ideal approach for development of an attenuated vaccine.
Flagella-less strains of other Borrelia pathogens such as Borrelia coriacei, which is associated with epidemic bovine abortion, Borrelia anserina, which causes avian spirochetosis, Borrelia recurrentis, Borrelia hermsii, Borrelia turicatae, Borrelia duttoni, Borrelia persica, and Borrelia hispanica, which cause relapsing fever, and any of a number of other Borrelia pathogens would possess similar advantages.